Functional luteinizing hormone (LH) receptors are critical to fertility in both males and females. These receptors are also important because of increasing evidence that naturally-occurring, constitutively active mutations of the LH receptor lead to precocious puberty in males and are implicated in the development of ovarian and testicular tumors. The overall goal of this research is to understand the role of luteinizing hormone receptor structure in regulation of male and female fertility. In Aim I we will determine whether isolated LH receptors form dimers or oligomers following hormone binding. Time-resolved phosphorescence anisotropy measurements of LH receptor rotational diffusion and fluorescence imaging of single receptors will be used. In Aim 2 we will explore the role of the LH receptor self-association in signaling. We will determine whether constitutively active LH receptors are self-associated and whether extensive crosslinking of LH receptors results in signaling. In Aim 3 we will ask whether LH receptors move into lipid rafts following binding of hormone and whether receptor self-association necessarily precedes this translocation. Raft isolation together with tracking of individual LH receptors on viable cells will be used to determine whether LH receptors become localized in small membrane microdomains following binding of hormone. In Aim 4 we will determine whether LH receptor desensitization occurs within membrane rafts. The isolation of membrane rafts at various times following receptor desensitization will establish whether there is orchestrated movement of LH receptors into and out of rafts. Our laboratory has developed laser optical instrumentation for cell membrane studies that are equaled by few other laboratories. The application of these instrumental methods will provide insight into membrane events accompanying LH-receptor mediated signal transduction and receptor desensitization. These studies will also draw on the experience of the PI as well as the expertise of Dr. Colin Clay, a member of the Animal Reproduction and Biotechnology Laboratory, and of Dr. Michael Tamkun, a neurobiologist at Colorado State University.